Surfactant-associated protein D (SP-D) is an important effector of antimicrobial host defense and innate immunity. Like Surfactant Protein A, SP-D shows specific interactions with conserved cell wall glycoconjugates and virulence factors expressed on the surfaces of a wide variety of important respiratory pathogens, including Gram-negative bacteria. The major goal of the project is to further define the structural determinants for the interactions of SP-D with Gram-negative lipopolysaccharides (LPS). We hypothesize that the SP-D-dependent recognition of LPS is primarily determined by interactions of the SP-D carbohydrate recognition domain with conserved, but variably modified, heptoses of the inner core oligosaccharide of LPS, or with specific mannose-rich repeating units of the 0-polysaccharide. These interactions, involve residues that flank the primary carbohydrate site, but are also influenced by the higher order oligomerization of trimeric subunits. In preliminary experiments, we observed species differences in the interactions of SP-D with specific forms of rough LPS in vitro. Guided by comparisons of primary sequence with the crystal structure of SP-D, we have begun to elucidate specific structural determinants of R-LPS recognition, and demonstrate the feasibility of modifying SP-D dependent interactions with R-LPS in vitro. For this application, we propose: 1) to characterize the mechanisms of interaction of SP-D with structurally defined forms of Gram-negative LPS, initially focusing on contributions of inner core heptose and heptose modifications; 2) to further define the contributions of LPS binding to the SP-D dependent recognition of genetically defined strains of Gram-negative bacteria; and 3) to elucidate the contributions of SP-D/LPS interactions to the regulation of the macrophage response using defined forms of LPS with known SP-D reactivity. To achieve these aims we will employ defined bacterial mutants with known SP-D reactivity and panels of well-characterized wild-type and mutant recombinant proteins. In addition we will pursue a variety of collaborative structural and biophysical studies that will allow us to better understand key interactions at the molecular level. Together, the proposed studies will yield important new information relating to the biologic activities of lung collectins and the response of the lung to gram-negative organisms, gram-negative LPS, and respiratory viruses. RELEVANCE (See instructions): Gram-negative bacteria are major causes of morbidity and mortality in hospital and community environments. This can largely be attributed to the toxic effects of their cell wall lipopolysaccharides (LPS). Surfactant Protein D (SP-D) plays important roles in the host response to LPS. The proposed studies will elucidate the mechanisms of interaction of SP-D and LPS, and further define the specific contributions of SP- D to the cellular response to LPS.